Major objectives of the proposed research are to isolate 100 mg of homogeneous dihydrofolate reductase (DHFR) from Pseudomonas aeruginosa, crystallize the enzyme and determine its three-dimensional structure using x-ray diffraction methods. Armed with the resulting detailed knowledge of the geometrical and topographical properties of Pseudomonas DHFR in relation to the known structure of the corresponding vertebrate enzyme, we will endeavor to design specific pharmacologically active inhibitors that selectively cripple the bacterial reductase without interfering with one-carbon metabolism in the host. It is intended that such novel inhibitors will address the current critical need for new anti-microbics with enhanced activity against Pseudomonas aeruginosa. Intermediate objectives of Phase I research are: 1) to isolate P. aeruginosa DHFR of sufficient purity and in sufficient quantity for biochemical characterization and for protein sequence determination; 2) to characterize the interaction of purified P. aeruginosa DHFR with the antifolate trimethoprim (TMP) in terms of absolute affinity and Ki for the DHF THF conversion; and 3) to clone the structural gene for P. aeruginosa DHFR.